The introduction of specific modifications in the prokaryotic and eukaryotic genome is a powerful tool in studying gene function both at the level of individual cells and in the context of a complete organism. In addition, modification of specific genes may result in the generation of industrially and medically important organisms, whereas correction of defective alleles in eukaryotic cells may provide a substantial step forward in the development of somatic gene-therapy protocols.
The method of genetic modification relies on the ability of virtually every cell type to exchange DNA sequences with a high degree of nucleotide sequence similarity by a process which is called homologous recombination (Kucherlapati R. et al., 1988). Briefly, the method of genetic modification involves the generation of a so-called targeting construct, a DNA sequence that is largely identical to the specific chromosomal locus to be modified, but differing from this locus by specific modifications. These modifications can be as small as the deletion, insertion or substitution of a single base-pair, or be as large as the deletion or insertion of ten's of kilobase pairs. On entry of the targeting construct into the cell, exchange of sequences flanking the modification with their chromosomal counterparts, will result in the introduction of the modification into the recipient chromosome.
The efficiency of homologous recombination in both prokaryotes and eukaryotes strongly depends on complete sequence identity of exchanging DNA strands (Schen, P. et al., 1986; Nassif, N., et al. 1993; Waldman, A. S. et al., 1988; Te Riele, H., 1992). Thus, sequence dissimilarities as small as 0.5% can already strongly impede homologous recombination. In several bacterial species, Escherichia coli, Salmonella typhimurium and Streptococcus pneumoniae as well as in the yeast Saccharomyces cerevisiae it has been unequivocally shown that the DNA mismatch repair system is responsible for suppressing recombination between homologous but non-identical DNA sequences (Rayssiguier, C. et al., 1989; Claverys, J. P. et al., 1986; Selva, E. et al., 1995).
Genetic Modification of Mice
The introduction of specific genetic alterations into the germ line of mice was made possible by a combination of new techniques in molecular biology and embryology that have become available during the last ten years (Capecchi, M. et al. 1989; Frohman, M. A. et al., 1989; Hogan, B. 1994). The method entails the introduction of a planned genetic modification in embryonic stem (ES) cells by homologous recombination. ES cells are originally derived from the inner cell mass of 3.5 day old pre-implantation embryos and can be maintained by in vitro culture as immortalized cell lines, retaining the undifferentiated state under appropriate culture conditions. At present, a number of ES cell lines are available. ES cells injected into the blastocoel of 3.5 day blastocyst-stage embryos can efficiently compete with the inner-cell-mass cells of the recipient blastocyst in embryonic development thus generating a chimeric mouse consisting of cells derived from the recipient blastocyst and the injected ES cells. Should the in vitro modified ES cells contribute to the germ line of chimeric animals, the ES cell genome can be transmitted to the next generation resulting with animals that carry the introduced modification in all their cells. Intercrossing of such animals reveals the phenotypic consequences of homozygosity of the modified gene.
The most commonly introduced genetic modification in ES cells is gene disruption leading to gene inactivation. This allows the study of gene function by analyzing the consequences of the absence of a particular gene in the context of a complete organism. Also, many hereditary human diseases result from homozygosity, i.e. presence of two defective alleles, of specific genes [e.g., cancer predisposition syndromes like hereditary nonpolyposis colorectal cancer (Modrich, P., 1994), Li Fraumeni syndrome (Malkin et al., 1990), retinoblastoma (Hansen, M. F., 1988)]. The generation of mouse models for such diseases by disruption of the mouse homologues of the genes involved provides an invaluable tool for studying hereditary diseases in an experimental setting.
Gene Disruption in ES Cells
The inactivation of a specific gene in ES cells via homologous recombination starts with the preparation of a DNA targeting construct (Thomas, K. R. et al., 1987). Two types of targeting constructs can be used. In a replacement-type vector, a drug-resistance marker gene (conferring to the cell resistance to drugs like G418, Hygromycin B, Puromycin, Histidinol) disrupts a sequence which is homologous to a sequence in or around the target gene in the recipient genome; in an insertion-type vector the marker gene flanks the homologous sequence. The targeting construct is introduced into ES cells by electroporation (or alternatively by Ca-Phosphate precipitation, liposome-mediated DNA transfer or micro-injection) and cells are selected for stable integration of the targeting construct into the recipient genome by growth in medium containing the appropriate drug. Drug-resistant colonies are the result of either one of two events: integration of the targeting construct at a random site in the genome or integration of the marker gene in the target locus via homologous recombination between the flanking sequences and their chromosomal counterparts. The replacement-type vector integrates by homologous recombination on both sides of the marker gene: the insertion-type vector integrates by a single homologous recombination event leading to duplication of the region of homology. Thus, the marker gene serves two purposes: it allows selection of cells that have taken up the targeting construct and, on integration via homologous recombination, it will disrupt the target gene thereby modifying its function.
Distinguishing ES cell clones resulting from homologous recombination from those resulting from random integration, requires the DNA of individual clones to be analyzed by Southern hybridization or the polymerase chain reaction.
Unfortunately, in many targeting experiments, random integration was often found to be far more efficient than homologous recombination and also large variations in targeting efficiency were observed for different genes (Camerini-Otero, R. D. et al. 1990). In this respect, mammalian cells differ from bacteria and lower eukaryotes like yeast (Sulston, J. E. et al., 1977), Leishmania major (Cruz., A., 1990) or Trypanosoma brucei (Ten Asbroek, A. et al., 1990), where integration of exogenous DNA into the recipient genome exclusively or predominantly occurs via homologous recombination. To date, three factors clearly affecting the recovery of homologous recombinants in mammalian cells have been identified. First, the frequency of homologous recombination increases substantially with the total length of the homologous sequences up to 14 kilobase pairs (Deng, C. et al., 1992). Second, the expression level of the marker gene at the target locus affects the frequency of recovery of homologous recombinants: low expression may lead to loss of homologous recombinants, whereas high expression may allow selection of homologous recombinants at an elevated drug concentration (Hanson, K. D. et al., 1993). Third, sequence dissimilarities between the targeting construct and the chromosomal target locus strongly suppress the efficiency of homologous recombination (Te Riele, H. et al., 1992).
High Efficiency Targeting with Isogenic DNA Constructs
The suppression of homologous recombination in ES cells by small sequence dissimilarities became clear by a gene targeting experiment aimed at disrupting the Retinoblastoma gene with a neomycin resistance marker gene. Two targeting constructs were prepared carrying the neomycin resistance marker embedded in 10.5 kb of Rb sequence. In one construct the Rb sequence was derived from an isogenic mouse strain 129, and was therefore identical to the corresponding chromosomal locus in the ES cells, which were also derived from mouse strain 129. In the other construct, the Rb sequence was derived from a non-isogenic mouse strain BALB/c (Te Riele et al., 1992; Table 1). The two constructs contained corresponding Rb sequences and were therefore largely similar. However, they differed approximately 0.6% at the nucleotide level (which corresponds to the level of sequence polymorphism found in the human population). Thus, in a stretch of 1687 base pairs that was sequenced, the BALB/c sequence differed from the 129 sequence by 9 base pair substitutions, three small deletions (of 1, 4 and 6 nucleotides) and two polymorphic CA-repeats (see Te Riele et al., 1992). On introduction of these constructs in 129-derived ES cells, homologous recombination at Rb with the 129-derived construct was 50-fold more efficient than with the nonisogenic BALB/c-derived construct. To provide additional evidence that the suppression of recombination was solely dependent on the polymorphisms between the endogenous locus and the targeting DNA, the inverse experiment was performed, i.e. targeting of a BALB/c-derived ES cell line with the 129- and BALB/c-derived constructs. This experiment yielded the inverse result, i.e. a higher targeting efficiency with the BALB/c-derived construct than with the nonisogenic 129-derived construct.
With a somewhat different targeting construct, consisting of a hygromycin resistance gene embedded in 17 kb of isogenic Rb DNA, we observed that 80% of all Hygromycin B-resistant colonies resulted from homologous recombination (Te Riele et al., 1992). This demonstrates that, in the presence of perfect homology, also in mammalian cells homologous recombination rather than random integration can be the predominant event.
Although clearly not all problems of gene targeting have been solved, many genes have now been successfully targeted by the use of isogenic DNA targeting constructs. However, genetic modification of cells derived from an outbred organism can become a difficult endeavor as isogenic targeting constructs are not easily available. In this case, efficient gene targeting would require the targeting construct to be prepared from DNA derived from the target cell. Especially in the context of gene therapy, this would raise a tremendous practical obstacle to correction of a defective gene. Also, base sequence divergence imposes a major barrier to exchanging a large chromosomal region of one species by the syntenic region of another species.
The Introduction of Subtle Mutations
A related problem exists in methodologies to introduce subtle mutations into the germ line of a transgenic animal. Although protocols for disruption of genes in inbred ES cell lines (and in somatic cell lines of which isogenic DNA targeting constructs can be prepared) are rather well developed, the introduction of more subtle mutations is not straightforward. Current protocols are variations on a two-step procedure in which first a marker gene is introduced into the target gene followed by replacement of the marker gene by the desired subtle mutation (Te Riele, H. et al., 1992). This procedure requires the marker gene to be selectable both for its presence (first step) and its absence (replacement by the subtle mutation).
Useful marker genes are the Hprt minigene to be used in Hprt-deficient ES cells (positive selection in HAT medium: negative selection by 6-thioguanine) and a combination of the neomycin resistance gene (positive selection by G418) and the Herpes Simplex Virus thymidine kinase gene (negative selection by Gancyclovir). In an alternative procedure, the subtle mutation and the marker gene are present on the same targeting construct and concomitantly introduced into the genome by homologous recombination. If an insertion-type vector was used, the marker gene can be removed by intrachromosomal recombination between the duplicated sequences that were generated during the first integration event (Hasty, P. et al., 1993). In case of a replacement-type vector, the marker gene can be removed if it was flanked by two site-specific-recombination sites (e.g. loxP sites). Recombination between these sites on introduction into the cell of the loxP-specific recombinase Cre will remove the marker gene from the genome (Kilby, N. et al., 1993).
Although either of the above mentioned procedures has allowed the subtle modification of a number of genes in ES cells, they are highly demanding as to the generation of appropriate DNA targeting constructs and the culturing of ES cells under various selective conditions.
Therefore, an attractive alternative to these procedures might be the use of small single- or double-stranded oligonucleotides (up to 100 bases or base pairs), which are identical to the target locus except for one or several base pair alterations. However, our finding that base sequence dissimilarities as small as 0.6% strongly suppress homologous recombination, may impose a major impediment to using such oligonucleotides for the introduction of subtle genetic modifications. There is thus a need in the art to allow the subtle modification of cell lines and cells derived from living organisms and temporarily cultured in vitro.